The present invention relates generally to chimeric polypeptides containing chemokine polypeptide domains. More specifically, the invention relates to the expression in host cells of recombinant polynucleotide sequences encoding chemokine polypeptides covalently attached to heterologous polypeptides, and the use of such chimeric polypeptides as research tools for identifying chemokine receptors, as vaccine adjuvants, as agents for the chemotactic recruitment of migratory cells, as agents for the stimulation or inhibition of angiogenesis, as agents against autoimmune diseases and inflammation, and as agents to inhibit the binding of HIV to certain receptors.
Chemokines (or chemotactic cytokines) are a class of cytokine molecules capable of chemotactically attracting migratory cells, and are involved in cell recruitment and activation in inflammation. Chemokines generally have small molecular weights in the range of 8-10 kDa and, like other small proteins such as cytokines, are believed to be rapidly inactivated in vivo, resulting in relatively short biological half-lives for these proteins. Most chemokines can be divided into two subgroups, CXC or CC, on the basis of the spacing of two highly-conserved cysteine amino acids near the amino terminus of these proteins. Within the CXC and CC subgroups, chemokines are further grouped into related families based on amino acid sequence similarity between them. CXC chemokine families include the IP-10 and Mig family; the GROxcex1, GROxcex2, and GROxcex3 family; the interleukin-8 (IL-8) family; and the PF4 family. CC chemokine families include the monocyte chemoattractant protein (MCP) family; the family including macrophage inhibitory protein-1xcex1 (MIP-1xcex1), macrophage inhibitory protein-1xcex2 (MIP-1xcex2), and regulated on activation normal T cell expressed (RANTES); and the lymphotactin family. The chemokines stromal cell-derived factor 1xcex1 (SDF-1xcex1) and stromal cell-derived factor 1xcex2 (SDF-1xcex2) form a chemokine family that is approximately equally related by amino acid sequence similarity to the CXC and CC chemokine subgroups. Individual members of the chemokine families are known to be bound by at least one chemokine receptor, with CXC chemokines generally bound by members of the CXCR class of receptors, and CC chemokines by members of the CCR class of receptors. For example, SDF-1xcex1 is known to be a ligand for the CXCR receptor fusin/CXCR4, and MIP-1xcex1 is bound by the CCR receptors CCR1, CCR4, and CCR5.
The presence of a chemokine gradient attracts migratory cells such as lymphocytes, leukocytes, and antigen-presenting cells (APCs) that may participate in autoimmune reactions, inflammation, or normal immune responses, or that may release other intercellular factors to stimulate or inhibit angiogenesis or other cellular processes. For example, the initiation of autoimmune disease requires the infiltration or recruitment of lymphocytes able to respond against self proteins into the organ bearing the antigenic self proteins. Inflammatory atherosclerotic lesions are due in part to infiltration of the vascular compartment by leukocytes recruited to the site. To induce an immune response, antigenic proteins and glycoproteins must bind to the surface of B lymphocytes to stimulate antibody production, and must be taken up by antigen-presenting cells, processed, and represented to T lymphocytes to mediate a T-lymphocyte response. Migratory cells that secrete IP10 or IL-8, when attracted by a chemokine gradient to a particular site, respectively may inhibit or stimulate the formation of blood vessels at that site. Chemokines may be used to establish a chemoattractive gradient for migratory cells that are expressing the appropriate chemokine receptors, or to obscure an existing chemoattractive gradient.
Chemokine receptors are also involved in functions other than chemotaxis, such as interacting with viral proteins. HIV-1 is known to bind to certain proteins on the surface of cells in order to gain entrance into these cells and replicate or integrate the viral gene into the host DNA. The CD4 protein on T lymphocytes and other cells, including certain antigen presenting cells, has been shown to be bound by the HIV-1 viral envelope protein gp120. This is believed to induce in gp120 a conformational change that then exposes regions of gp120 and perhaps CD4 that subsequently bind to a chemokine receptor. To date CXCR4 (also known as fusin), CCR5, and several other chemokine receptors have been identified as co-receptors for HIV-1. Monocyte-tropic (M-tropic) isolates of HIV-1 require interaction with CCR5 in order to infect cells, while T-lymphocyte-tropic (T-tropic) HIV-1 isolates require another coreceptor, CXCR4, for infection. There is some evidence indicating that HIV-1 can also use other CCR receptors such as CCR2 and CCR3 to gain entry into cells expressing them. For some HIV-2 isolates, it appears that certain chemokine receptors such as fusin/CXCR4 alone can provide the cell-surface protein needed for binding and entrance into the cell.
HIV-1 injection of cells expressing CD4 and fusin/CXCR4 is greatly decreased by the addition of purified SDF-1xcex1, which is bound by fusin/CXCR4. We have found that preincubation of cells in the presence of purified SDF-1xcex1 for a short period of time at 37xc2x0 C. causes a profound down-regulation of the receptor. This down-regulation of fusin/CXCR4 correlates with a decrease in the ability of HIV-1 to infect cells.
There is a continuing requirement for new compositions that will enhance, alter, or inhibit the effects of chemokine-receptor interactions, and for methods for their use.
Applicants have for the first time constructed novel chimeric DNA molecules encoding chimeric polypeptides comprising chemokine polypeptide domains. Chimeric polypeptides expressed from these constructs have exhibited novel properties, including novel interactions with cells expressing chemokine receptors.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, the chimeric polypeptide comprising at least one chemokine polypeptide covalently attached to at least one heterologous polypeptide. Preferably, the chemokine polypeptide is SDF-1xcex1, MIP-1xcex1, or MIP-1xcex2, or is derived from SDF-1xcex1, MIP-1xcex1, or MIP-1xcex2. Preferably, the heterologous polypeptide is an Fc polypeptide.
Another embodiment provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein a heterologous polypeptide is covalently attached to the amino terminus of a chemokine polypeptide, preferably by a linker polypeptide.
Another embodiment provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein a heterologous polypeptide is covalently attached to the carboxyl terminus of a chemokine polypeptide, preferably by a linker polypeptide.
In another embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 from nucleotide 12 to nucleotide 1213;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 from nucleotide 69 to nucleotide 1213;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 from nucleotide 72 to nucleotide 1213;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2 from nucleotide 75 to nucleotide 1213;
(e) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:2;
(f) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone S1-3 deposited under accession number ATCC XXXXX;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone S1-3 deposited under accession number ATCC XXXXX;
(h) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:1;
(i) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:1 from amino acid 20 to amino acid 328;
(j) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:1 from amino acid 22 to amino acid 328;
(k) a polynucleotide encoding a chimeric polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:1;
(l) a polynucleotide comprising a nucleotide sequence complementary to any one of the polynucleotides specified in (a)-(k) above; and
(m) a polynucleotide capable of simultaneously hybridizing under stringent conditions to sequences encoding the chemokine polypeptide and to sequences encoding the heterologous polypeptide in any one of the polynucleotides specified in (a)-(l) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:2 from nucleotide 12 to nucleotide 1213; the nucleotide sequence of the full-length protein-coding sequence of clone S1-3 deposited under accession number ATCC XXXXX; or the nucleotide sequence of the mature protein-coding sequence of clone S1-3 deposited under accession number ATCC XXXXX.
In a further embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 12 to nucleotide 1207;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:4 from nucleotide 69 to nucleotide 1207;
(c) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:4;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone SK2-2 deposited under accession number ATCC XXXXX;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone SK2-2 deposited under accession number ATCC XXXXX;
(f) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:3;
(g) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:3 from amino acid 20 to amino acid 326;
(h) a polynucleotide encoding a chimeric polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:3;
(i) a polynucleotide comprising a nucleotide sequence complementary to any one of the polynucleotides specified in (a)-(h) above; and
a polynucleotide capable of simultaneously hybridizing under stringent conditions to sequences encoding the chemokine polypeptide and to sequences encoding the heterologous polypeptide in any one of the polynucleotides specified in (a)-(i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:4 from nucleotide 12 to nucleotide 1207; the nucleotide sequence of the full-length protein-coding sequence of clone SK2-2 deposited under accession number ATCC XXXXX; or the nucleotide sequence of the mature protein-coding sequence of clone SK2-2 deposited under accession number ATCC XXXXX.
In another embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 15 to nucleotide 1225;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 81 to nucleotide 1225;
(c) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:6;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone MP-1 deposited under accession number ATCC XXXXX;
(e) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone MP-2 deposited under accession number ATCC XXXXX;
(f) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone MP-6 deposited under accession number ATCC XXXXX;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone MP-1 deposited under accession number ATCC XXXXX;
(h) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone MP-2 deposited under accession number ATCC XXXXX;
(i) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone MP-6 deposited under accession number ATCC XXXXX;
(j) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:5;
(k) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:5 from amino acid 23 to amino acid 331;
(l) a polynucleotide encoding a chimeric polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:5;
(m) a polynucleotide comprising a nucleotide sequence complementary to any one of the polynucleotides specified in (a)-(l) above; and
(n) a polynucleotide capable of simultaneously hybridizing under stringent conditions to sequences encoding the chemokine polypeptide and to sequences encoding the heterologous polypeptide in any one of the polynucleotides specified in (a)-(m) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:6 from nucleotide 15 to nucleotide 1225; the nucleotide sequence of the full-length protein-coding sequence of clones MP-1, MP-2, and MP-6 deposited under accession numbers ATCC XXXXX, ATCC XXXXX, and ATCC XXXXX, respectively; or the nucleotide sequence of the mature protein-coding sequence of clones MP-1, MP-2, and MP-6 deposited under accession numbers ATCC XXXXX, ATCC XXXXX, and ATCC XXXXX, respectively.
In a further embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a chimeric polypeptide, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 16 to nucleotide 1226;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:8 from nucleotide 85 to nucleotide 1226;
(c) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:8;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein-coding sequence of clone MPB-X deposited under accession number ATCC XXXXX;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein-coding sequence of clone MPB-X deposited under accession number ATCC XXXXX;
(f) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:7;
(g) a polynucleotide encoding a chimeric polypeptide comprising the amino acid sequence of SEQ ID NO:7 from amino acid 24 to amino acid 331;
(h) a polynucleotide encoding a chimeric polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:7;
(i) a polynucleotide comprising a nucleotide sequence complementary to any one of the polynucleotides specified in (a)-(h) above; and
(j) a polynucleotide capable of simultaneously hybridizing under stringent conditions to sequences encoding the chemokine polypeptide and to sequences encoding the heterologous polypeptide in any one of the polynucleotides specified in (a)-(i) above.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:8 from nucleotide 16 to nucleotide 1226; the nucleotide sequence of the full-length protein-coding sequence of clone MPB-X deposited under accession number ATCC XXXXX; or the nucleotide sequence of the mature protein-coding sequence of clone MPB-X deposited under accession number ATCC XXXXX.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, preferably a mammalian cell, transformed with such polynucleotide compositions.
Processes are also provided for producing a chimeric polypeptide, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The polypeptide produced according to such methods is also provided by the present invention. Preferred embodiments include those in which the polypeptide produced by such process is a mature form of the polypeptide.
In other embodiments, the present invention provides a composition comprising a chimeric polypeptide, the chimeric polypeptide comprising at least one chemokine polypeptide covalently attached to at least one heterologous polypeptide. Preferably, the chemokine polypeptide is SDF-1xcex1, MIP-1xcex1, or MIP-1xcex2, or is derived from SDF-1xcex1, MIP-1xcex1, or MIP-1xcex2. Preferably, the heterologous polypeptide is an Fc polypeptide.
A further embodiment provides a composition comprising a chimeric polypeptide, wherein a heterologous polypeptide is covalently attached to the amino terminus of a chemokine polypeptide, preferably by a linker polypeptide.
Another embodiment provides a composition comprising a chimeric polypeptide, wherein a heterologous polypeptide is covalently attached to the carboxyl terminus of a chemokine polypeptide, preferably by a linker polypeptide.
In another embodiment, the present invention provides a composition comprising a chimeric polypeptide, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:1;
(b) the amino acid sequence of SEQ ID NO:1 from amino acid 20 to amino acid 328;
(c) the amino acid sequence of SEQ ID NO:1 from amino acid 21 to amino acid 328;
(d) the amino acid sequence of SEQ ID NO:1 from amino acid 22 to amino acid 328; and
(e) fragments of the amino acid sequence of SEQ ID NO:1.
Preferably, such chimeric polypeptide comprises the amino acid sequence of SEQ ID NO:1.
In a further embodiment, the present invention provides a composition comprising a chimeric polypeptide, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:3;
(b) the amino acid sequence of SEQ ID NO:3 from amino acid 20 to amino acid 326; and
(c) fragments of the amino acid sequence of SEQ ID NO:3.
Preferably, such chimeric polypeptide comprises the amino acid sequence of SEQ ID NO:3.
In another embodiment, the present invention provides a composition comprising a chimeric polypeptide, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:5;
(b) the amino acid sequence of SEQ ID NO:5 from amino acid 23 to amino acid 331; and
(c) fragments of the amino acid sequence of SEQ ID NO:5.
Preferably, such chimeric polypeptide comprises the amino acid sequence of SEQ ID NO:5.
In a further embodiment, the present invention provides a composition comprising a chimeric polypeptide, wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:7;
(b) the amino acid sequence of SEQ ID NO:7 from amino acid 24 to amino acid 331; and
(c) fragments of the amino acid sequence of SEQ ID NO:7.
Preferably, such chimeric polypeptide comprises the amino acid sequence of SEQ ID NO:7.
Polypeptide compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such polypeptide are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.
The present invention also provides methods for identifying molecules capable of interacting with a chimeric polypeptide which comprise:
(a) combining a composition of claim 23 with a composition comprising molecules to be tested for interaction, forming a first mixture;
(b) combining the first mixture with a composition comprising indicator molecules, so that the indicator molecules are capable of being altered by the first mixture; and
(c) detecting the presence of altered indicator molecules.
Methods are also provided for attracting migratory cells to a region of an organism which comprises administering therapeutically effective amounts of at least one composition comprising a chimeric polypeptide.
Methods for stimulating or inhibiting angiogenesis, which comprise administering therapeutically effective amounts of at least one composition comprising a chimeric polypeptide, are also provided.
Methods are also provided for preventing, treating, or ameliorating an inflammatory or an autoimmune condition, which comprise administering therapeutically effective amounts of at least one composition comprising a chimeric polypeptide.
Methods for enhancing antigen-presenting cell activity, which comprise administering therapeutically effective amounts of at least one composition comprising a chimeric polypeptide, wherein at least one chimeric polypeptide comprises antigen molecules, are also provided.
Methods are provided for inducing an immune response which comprise administering a vaccine and therapeutically effective amounts of at least one composition comprising a chimeric polypeptide.
Methods for altering receptor function which comprise causing a receptor to bind at least one chimeric polypeptide, and for decreasing receptor function which comprise causing a receptor to bind at least one chimeric polypeptide, resulting in a decrease in the number of functional receptor molecules, are provided.
Methods are provided for preventing, treating, or ameliorating HIV infection which comprise administering therapeutically effective amounts of at least one composition comprising a chimeric polypeptide. Preferably, the chemokine polypeptide of the chimeric polypeptide comprises SDF-1xcex1 , MIP-1xcex1, or MIP-1xcex2.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.